|Year : 2020 | Volume
| Issue : 4 | Page : 550-556
|Effects of chitosan oligosaccharide and calcium hypochlorite on E. Faecali dentinal biofilm and smear layer removal - SEM analysis
Gurveen Kaur1, T Vinay Kumar Reddy1, Kondas Vijay Venkatesh1, Krishnan Mahalakshmi2
1 Department of Conservative Dentistry and Endodontics, SRM Kattankulathur Dental College and Hospital, SRM Institute of Science and Technology, Chennai, Tamil Nadu, India
2 Department of Microbiology, Sree Balaji Dental College and Hospital, BIHER, Bharath University, Chennai, Tamil Nadu, India
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|Date of Submission||08-Apr-2020|
|Date of Decision||29-Apr-2020|
|Date of Acceptance||10-Jun-2020|
|Date of Web Publication||16-Oct-2020|
| Abstract|| |
Background: The complex structure and irregularities of root canal walls are liable for infection by several bacterial species. Thus, the use of irrigants and auxiliary chemical solutions associated with instrumentation is necessary for effective eradication of the biofilm as well as complete removal of the smear layer. Aim: To evaluate the effects of calcium hypochlorite and chitosan oligosaccharide (COS) in disinfecting Enterococcus faecalis root canal biofilm and smear layer removal with minimal erosion. Materials And Methods: A total of 70 mandibular premolars were decoronated at the cementoenamel junction. The samples were biomechanically prepared, sterilized in an autoclave, and incubated with E. faecalis (ATCC-29212) bacteria for 21 days. Cleaning and shaping were done till maximum apical file size of #45 K. Specimens were randomly divided into 4 groups: GROUP I: Control Group, GROUP II: 5% Sodium Hypochlorite (NaOCl) solution followed by 17% EDTA solution, GROUP III: 5% Calcium Hypochlorite [Ca(OCl)2] solution followed by 17% EDTA solution and GROUP IV: 5% Ca(OCl)2 solution followed by 1% COS. The samples were subjected to microbial count followed by smear layer removal under scanning electron microscope (SEM) at coronal, middle and apical third. Statistical Analysis Used: Kruskal–Wallis Test and post-hoc Scheffe's test. Results: It was observed that Group IV showed the lowest amount of CFU count/mL and the highest amount of smear layer removal with a statistically significant difference (P < 0.05) when compared with the other three Groups. Conclusion: 5% Ca(OCl)2 solution with 1% COS solution effectively removed the Enterococcus faecalis biofilm and smear layer from the root canals with minimal erosion.
Keywords: Calcium hypochlorite, chitosan oligosaccharide, Enterococcus faecalis, smear layer
|How to cite this article:|
Kaur G, Kumar Reddy T V, Venkatesh KV, Mahalakshmi K. Effects of chitosan oligosaccharide and calcium hypochlorite on E. Faecali dentinal biofilm and smear layer removal - SEM analysis. Indian J Dent Res 2020;31:550-6
|How to cite this URL:|
Kaur G, Kumar Reddy T V, Venkatesh KV, Mahalakshmi K. Effects of chitosan oligosaccharide and calcium hypochlorite on E. Faecali dentinal biofilm and smear layer removal - SEM analysis. Indian J Dent Res [serial online] 2020 [cited 2020 Oct 31];31:550-6. Available from: https://www.ijdr.in/text.asp?2020/31/4/550/298403
| Introduction|| |
The principal objective of cleaning and shaping of the root canal system is to eradicate periradicular pathosis of pulpal origin and retain the natural tooth in its function and aesthetics. Various factors like anatomical variations in the root canal, presence of extra-radicular and intra-radicular infections, reaction to the foreign body, presence of cysts with cholesterol crystals lead to the persistence of a periradicular pathosis.Enterococcus faecalis is the most common cause of the persistence of periapical infection. Thus, the elimination of bacterial biofilm assumes a greater significance before root canal filling and is performed via mechanical instrumentation along with chemical irrigation.
Irrigation has a vital role in root canal treatment, which aids in removing bacteria, remnants of pulp tissue and dentinal debris from the root canal wall during instrumentation. Till date, there is not a single irrigating solution that alone satisfies all the objectives of irrigation. Thus, ideal irrigation should incorporate two or more irrigants, followed in a specific protocol.
The gold standard in irrigation is Sodium hypochlorite (NaOCl). However, NaOCl does not eliminate the smear layer and needs to be combined with 17% Ethylene Diamine Tetra Acetic acid (EDTA) to eliminate the inorganic debris during mechanical instrumentation. NaOCl along with EDTA has a cytotoxic potential and can lead to local tissue necrosis if extruded into the periapical area.,
Because of the limitations of the conventional irrigation protocols, an alternative approach is required to render the root canals pathogen-free. One such approach is the use of Calcium Hypochlorite [Ca(OCl)2] and a biopolymer such as Chitosan Oligosaccharide (COS) for disinfection of the root canals. Ca(OCl)2 is an industrial chemical available in granular form, which is used for disinfection, bleaching and decontamination treatment of water.
Chitosan [poly (1,4-b-D-glucopyranosamine)] is a naturally available polysaccharide with biodegradability, compatibility, low toxicity and bio-adhesion properties. Chitosan has broad-spectrum antibacterial property, high chelating effect and similarity in structure with extracellular matrix components.
Thus the aim of the study was to determine the antimicrobial efficacy of COS and Ca(OCl)2 against Enterococcus faecalis biofilm and evaluate the amount of smear layer removed with least dentinal erosion.
| Materials and Methods|| |
Selection and preparation of the samples
The study was approved under Ethical Clearance Number: 1256/IEC/2017. Seventy extracted single-rooted human mandibular premolars were selected for the study. Carious teeth, teeth with calcified and curved roots were excluded from the study. The debris and calculus were cleaned from the samples using ultrasonic scaling and were stored in physiological saline. The samples were decoronated at cementoenamel junction (CEJ) using a diamond disk to obtain a uniform root length of 17 mm.
The working length of the samples was determined using #15 K file size (Mani, Vietnam) and confirmed with radiovisiography (SATELEC X-mind, Acteon, Italy). The working length was considered 1 mm short of the apex (Ingles method of working length determination). Cleaning and shaping of root canals were done using step-back technique till apical enlargement of # 35 K file size (Mani, Vietnam). The canals were irrigated using a 5% NaOCl solution (Nimai Dento, India) and 17% EDTA solution (Meta Biomed, Republic of Korea) using side vented needles (Orikam). The sectioned samples were sterilized in an autoclave at a temperature of 121°C at 15 lbs pressure for 20 min to prevent contamination of the samples.
Cultivation and inoculation of E. faecalis biofilm
The sterile tooth samples were immersed in 5 mL of Mueller Hinton Broth (MHB) (Himedia Laboratories Pvt. Limited, Mumbai-India) in each test tubes and sterilization was repeated by plugging the test tubes with cotton. To obtain a cell density of 1.5 × 10-8 CFU/mL Enterococcous faecalis strains (ATCC 29212) culture was adjusted to 0.5 McFarland standards. The MHB containing the tooth samples were inoculated with 10 μL of Enterococcous faecalis strains (ATCC 29212) and were incubated for 3 weeks in an orbital shaker for biofilm formation. Every alternate day Mueller–Hinton culture broth was replaced to avoid nutrient depletion and accumulation of toxic end products.
Preparation of the solutions
The freshly prepared solutions of calcium hypochlorite and chitosan were used in this study. For the preparation of 5% Ca(OCl)2 solution, 5 gm of Ca(OCl)2 granules (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) were weighed on a precision balance and mixed with 100 mL of deionized water. The solution was prepared under constant agitation until all the granules were dissolved. For the preparation of 1% chitosan solution, 1 gm of chitosan oligosaccharide powder (Sisco Research Laboratories Pvt. Ltd. Mumbai India) was weighed and mixed with 100 mL of deionized water. The solution was mixed using a magnetic stirrer in order to get a homogenous clear solution.
Sample preparation for experimental groups
The sectioned tooth samples were inoculated and incubated with Enterococcous faecalis for 21 days. The samples were washed with normal saline and randomly distributed into four groups as per the irrigation protocol.
The canals were further cleaned and shaped using the Step-Back technique till #45K file size (Mani, Vietnam). During the instrumentation tooth samples were irrigated according to the groups distributed.
- GROUP I (Control): Specimens were irrigated with Saline (n = 10)
- GROUP II: Specimens were irrigated with 5% NaOCl solution and 17% EDTA solution (n = 20)
- GROUP III: Specimens were irrigated with 5% Ca(OCl)2 solution and 17% EDTA solution (n = 20)
- GROUP IV: Specimens were irrigated with 5% Ca(OCl)2 solution and 1% Chitosan solution (n = 20)
The specimens were irrigated with 2 mL of each irrigant for 5 min. Saline irrigation was done in between each irrigant to prevent the irrigant interactions and final irrigation was done with 5 mL of saline in all the samples.
Evaluation of antimicrobial activity
After following different irrigation protocols, #45 size sterile paper points (Dentsply) were placed in the root canals for 30 s following which the paper points were transferred to microcentrifuge tubes containing 1 mL of sterile normal saline. The microcentrifuge tubes were labeled and numbered according to the respective groups. A lawn culture was performed on Mueller Hinton Agar (MHA) with ten microliters of normal saline containing the paper points. The incubation of MHA plates was done at 37°C for 24 h. To calculate the bacterial load present in the samples, colony-forming unit (CFU) count was determined after 24 h of incubation with the help of a Digital Colony Counter.
Evaluation of smear layer removal
After the microbial count, the samples were split longitudinally in a buccolingual plane with a slow speed diamond disc without penetrating the canal to facilitate their vertical splitting. All the samples were split into two halves using a surgical chisel and mallet. The sectioned samples were mounted on stubs, gold- sputtering was done and examined under SEM (FEI Quanta, USA). Gold sputtering was done to prevent the charging of the specimen due to the accumulation of static electric fields. Representative areas were marked for each tooth specimen at the apical, middle and coronal third of the root. The images were taken at 3000 × magnifications for evaluation of absence or presence of smear layer [Figure 1], [Figure 2], [Figure 3].
|Figure 1: SEM images of Coronal third of root canal at 3000 × for Group I, II, III and IV|
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|Figure 2: SEM images of Middle third of root canal at 3000 × for Group I, II, III and IV|
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|Figure 3: SEM images of Apical third of root canal at 3000× for Group I, II, III and IV|
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The samples were coded in a blind manner based on a three-grade scale given by Torabinejad et al. (2003) to evaluate the degree of smear removal and dentinal erosion.
Score for smear layer removal
1: Presence of no smear layer on the root canals surfaces, and all the dentinal tubules were open and clean.
2: Presence of moderate smear layer i.e., smear layer absent on the root surface but present on the dentinal tubules.
3: Presence of heavy smear layer with smear layer covering the root canal surfaces and the dentinal tubules.
Score for dentin erosion
1: No erosion characterized by all the dentinal tubules looking normal in size and appearance.
2: Moderate erosion characterized by the presence of eroded peritubular dentin.
3: Severe erosion characterized by the destruction of intertubular dentin and connected with each other.
All the values recorded during the study were analyzed with statistical software IBM.SPSS 19.0 Version. Descriptive statistics, Mean and Standard error of mean were used to describe the results. To find the significant difference between the groups, non-parametric Kruskal–Walli's test was done followed by post hoc Scheffe's test for intergroup comparisons. The significance level was set as P < 0.05.
| Results|| |
Evaluation of antimicrobial efficacy
The highest bacterial load was seen in Group I with a mean count of 140900 CFU/mL, whereas Group IV significantly exhibited the lowest mean bacterial count with 15 CFU/mL (P-value < 0.05) in [Table 1].
[Table 2] shows inter-group comparison where Group I showed a high significant difference (P-value < 0.05) when compared to the other three groups.
Evaluation of smear layer removal
The sectioned tooth samples were observed under scanning electron microscope (SEM), and images were taken from coronal, middle and apical third of the root at 3000 × magnifications for evaluation of absence or presence of smear layer. The highest amount of smear layer was seen in Group I (Control Group) while the lowest amount was observed in Group IV in the coronal, middle and apical third region [Table 3]. On inter-group comparison of Group IV with the other three groups statistically high significant differences were observed between efficacies of smear layer removal [Table 4].
Evaluation of dentine erosion
The final parameter taken into consideration was the amount of dentinal erosion seen during irrigation. [Table 5] shows the mean amount of erosion seen in all the four groups at coronal, middle and apical third of the root canal. The lowest dentinal erosion with significant value (P = 0.0001) was seen in Group I and Group IV, whereas the highest value was obtained in Group II. Multiple comparison by post hoc Scheffe's test shows that Group I and Group IV when compared with the other two groups had a significant difference in dentin erosion [Table 6].
From the above results, it is evident that the highest antibacterial activity, as well as smear layer removal with minimal dentin erosion, was observed when the samples were irrigated with 5% Ca(OCl)2 solution followed by 1% chitosan solution. Samples irrigated with 5% NaOCl solution and 17% EDTA solution showed dentinal erosion as evident from SEM images in [Figure 1] and [Figure 3] (Group II).
| Discussion|| |
The root canal system has a complex nature due to the presence of many lateral and accessory canals, cul-de-sacs, abundant dentinal tubules, which are invaded by the bacteria during infection and smear layer formation in the course of mechanical instrumentation. These factors act as major hurdles in achieving complete debridement of root canal systems. It is imperative for an irrigant to eradicate microorganisms and smear layer from the root canal walls with least toxicity. The presence of a smear layer inhibits penetration of intracanal medicaments and the adaptation of sealer to the radicular dentin walls.
Bystrom and Sundqvist et al. assessed the antimicrobial efficacy of irrigants with mechanical instrumentation. They found that the bacterial colony count showed a positive culture after irrigation with saline. When irrigant like NaOCl was used alone or combined with EDTA collectively, the bacterial load was significantly reduced. Pashley et al. in the year 1985 demonstrated the toxic effects of NaOCl on vital tissues, causing hemolysis, necrosis and skin ulceration. Apart from this EDTA has little or no antibacterial effect and is cytotoxic to fibroblasts. Prolonged exposure of EDTA can lead to excessive removal of intratubular and peritubular dentin. NaOCl decreases the fracture resistance of the teeth, and affects the bond strength of adhesive restorations to dentin. Because of the harmful effects of these irrigants, alternative irrigants are being developed.
In this study, antimicrobial efficacy against Enterococcus faecalis, as well as smear layer removal was assessed using Ca(OCl)2 and COS. It was found that the maximum amount of smear layer removal and highest antimicrobial efficacy was observed when the root canals were irrigated with 5% Ca(OCl)2 solution followed by 1% Chitosan solution.
Enterococcus faecalis is gram-positive, facultative anaerobic cocci responsible for secondary infection and is a major cause for persisting root canal infections., For bacterial incubation, 21 days were considered for a structured biofilm formation and disinfection, thus simulating the clinical conditions in a better way. The choice of culture media was Mueller–Hinton Broth as it is readily available and commonly used for E. faecalis.
Ca(OCl)2 is a stable chemical material, which is commonly used for disinfection, bleaching and decontamination of water. It has antimicrobial activity and organic tissue- dissolving ability similar to NaOCl. However, it has 65% more available chlorine than NaOCl, and its concentration can be more accurate when incorporated into water, than diluting a concentrated solution. Ca(OCl)2 is available in the form of granules. In aqueous solution, it ionizes into hypochlorous acid and calcium hydroxide and the following reaction occurs:
Ca(OCl)2+2 H2O → 2 HOCl+Ca (OH)2
The dissolution of bovine tissues was similar when 5.25% NaOCl and 5% or 10% of Ca(OCl)2 was used. Ca(OCl)2 when accompanied with an ultrasonic irrigation system was effective in the eradication of microorganisms and can be helpful in bio-mechanical preparation of the root canals. It can be considered as a potential root canal irrigant. Conversely, it cannot be used as an intra-canal medicament because of the presence of chlorine ions, which can cause symptoms if extruded into the periapical tissues or can weaken the root structure.
Chitosan [β (1,4) D-glucosamine], is an abundant natural positively charged, hydrophilic marine biopolymer obtained from chitin, which is the major source of the exoskeleton of crustaceans like crabs, shrimps and rarely fungi. It is synthesized by a process known as deacetylation and is available in different forms including beads, powder, flakes, films, hydrogels and nanoparticles. Chitosan readily dissolves in acidic medium but is insoluble in solutions where pH is greater than 6.5 due to the presence of the remaining amino group. Further, chitosan has the capacity to chelate with heavy metal ions.
In this study, chitosan oligosaccharide (COS) was used, which is an oligomeric derivative of β-(1,4) D-glucosamine. COS is soluble in water, unlike the chitosan. It is also non-cytotoxic, biocompatible material and is quickly absorbed through the intestine and excreted in the urine. The structure of chitosan is similar to the extracellular matrix components, and it reinforces collagen by enzymatic degradation.
Chitosan has exceptional antimicrobial properties against bacterial, fungi and viruses. It is more effective for gram-positive than gram-negative bacteria. Three mechanisms have been put forward for antimicrobial activity. These are: i) the surface interaction of ions causing cell wall leakage; ii) by inhibiting protein and mRNA production in the bacterial nucleus, and finally iii) chelation of metal ions, forming an external barrier and inhibiting essential nutrients for the growth of microbes. The most accepted mode of action is the interaction between the positively charged amino groups of chitosan to the negatively charged bacterial cell membrane and changing the cell permeability.
The therapeutic effect of chitosan was demonstrated in a study by Muzzarelli et al. who reported an inhibitory, bactericidal and fungicidal activity by N-carboxybutyl chitosan against 298 cultures of various pathogens.
Chitosan also has a chelating effect on heavy metals suggested by two theories. The first theory suggests that the ability of chitosan to chelate is through the presence of two or more amino groups that interact with the same metal ion. This is referred to as bridge model. The second theory is known as the pendant model, which emphasises that one amino group is used in the binding, and the metal ion is attached to the amino group like a pendant. The removal of the smear layer from dentin can be due to either of the two theories.
Since chitosan has both antibacterial and chelating effect, the samples irrigated with calcium hypochlorite and chitosan showed significant eradication of E. faecalis. The use of calcium hypochlorite followed by chitosan showed a synergistic effect. For irrigation, side vented tips were used as the hydrodynamic activation of irrigant prevented extrusion of the irrigant beyond the apex.
Aysin Dumani et al. concluded that calcium hypochlorite is equally effective in removing Enterococcus faecalis from the radicular dentin when compared with sodium hypochlorite. Natalia Gomes Leonardo et al. evaluated the chlorine content available, pH and surface tension of Ca(OCl)2 and NaOCl. It was observed that more chlorine content was liberated along with greater surface tension property due to the high alkalinity of Ca(OCl)2 as compared to NaOCl. Kamble AB et al. compared 0.2% chitosan with 17% EDTA in removing the smear layer. He concluded that 0.2% chitosan was efficient in removing the smear layer from the apical third region of the root canal.
The limitation of the study was that it was not feasible to assess the depth of penetration of E. faecalis in dentinal tubules and viability of this microorganism. Since it is an in-vitro study, the results cannot be extrapolated completely to in-vivo situations. Hence in order to draw the benefits and consequences to human beings further research may be required to evaluate the properties of root canal irrigants in detail.
| Conclusion|| |
Within the limitations of this study, it was concluded that 5% calcium hypochlorite solution when used in conjunction with 1% chitosan oligosaccharide solution in an irrigation protocol, was able to eliminate E. faecalis along with complete smear layer removal with least dentin erosion. The use of calcium hypochlorite with chitosan oligosaccharide can be considered as an alternative to conventional irrigants.
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Conflicts of interest
There are no conflicts of interest.
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] [Full text]
Dr. Gurveen Kaur
House No. 3113, Sector 35-D, Chandigarh 160022
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]
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